The objective of this project is to identify the events triggered by binding of phorbol diesters to their specific binding sites which actually lead to tumor promotion rather than epiphenomena. The roles of membrane lipid changes, reactive oxygen and monovalent cation transport have been probed and each has been found to be promotion relevant. Inhibitors of the phospholipid methyltransferase system, 3 DZA and SAH can block TPA-induced promotion in JB6 cells. H202 alone is a known promoter in vivo and in our in vitro system. Catalase preparations have abrogated TPA-induced changes in morphology, procollagen secretion and promotion itself. Arginine vasopressin, which enhances the mitogenic effect of several growth factors by inducing sodium influx, has induced anchorage independent growth. In each case, studies are in progress to definitively relate the expected mediators to promotion and to each other.